The Therapeutic Potential of Amniotic Fluid-Derived Stem Cells on Busulfan-Induced Azoospermia in Adult Rats



Busulfan is an alkylating chemotherapeutic agent that is routinely prescribed for leukemic patients to induce myelo-ablation. However, it also results in azoospermia and infertility in cancer survivors. This research was constructed to explore the possible therapeutic role of amniotic fluid-derived stem cells (AFSCs) in improving busulfan-induced azoospermia in adult rats.


Forty two adult male albino rats were randomized into: (1) control group, (2) azoospermia group, (3) spontaneous recovery group, and (4) AFSCs-treated group, in which AFSCs were transplanted through their injection into the testicular efferent ducts. The assessment included a histo-pathological examination of the seminiferous tubules by the light and transmission electron microscopes. Additionally, the confocal laser scanning microscope was used for confirmation of homing of the implanted cells. Moreover, we conducted an immuno-fluorescence study for detection of the proliferating cell nuclear antigen (PCNA) in the spermatogenic cells, epididymal sperm count, and a histo-morphometric study.


AFSCs successfully homed over the basement membrane of the injured seminiferous tubules. They greatly attenuated busulfan-induced degenerative and oxidative changes. They also caused a re-expression of PCNA in the germ cells, leading to resumption of spermatogenesis and re-appearance of spermatozoa.


AFSCs could be a promising treatment modality for male infertility induced by chemotherapy, as they possess prominent regenerative, anti-apoptotic, and anti-inflammatory potentials.

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Authors acknowledge the technical help at the Center of Excellence for Research in Regenerative Medicine Applications (CERRMA), Alexandria Faculty of Medicine. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author information




HFI carried out the experimental work, designed the experiments, and wrote the manuscript. SHS and TMZ shared in the experimental design, performed the analytical part of the study, supervised the research, and revised the manuscript. KFEM and AYM optimized the experimental design and gave a conceptual advice. All authors read and approved the manuscript.

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Correspondence to Heba F. Ibrahim.

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The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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The animal study was performed after receiving approval of the Research Ethics Committee for care and use of laboratory animals, approval No. 0201162, IRB code 00012098, FWA code 00018699, membership of Alexandria University in the International Council of Laboratory Animal Science organization (ICLAS-

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Ibrahim, H.F., Safwat, S.H., Zeitoun, T.M. et al. The Therapeutic Potential of Amniotic Fluid-Derived Stem Cells on Busulfan-Induced Azoospermia in Adult Rats. Tissue Eng Regen Med 18, 279–295 (2021).

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  • Amniotic fluid-derived stem cells
  • Busulfan
  • Azoospermia
  • Efferent ducts